蜜环菌遗传转化体系的构建及应用

	蜜环菌遗传转化体系的构建及应用
	粟忠祥
导师	吴建强
关键词	蜜环菌,遗传转化,转基因技术,RNAi Armillaria,Genetic transformation,Transgenic technology,RNAi
摘要	蜜环菌属（Armillaria spp.）真菌隶属于担子菌纲（Basidiomycetes）蘑菇目（Agaricales）膨瑚菌科（Physalacriaceae），是一个世界性广泛分布的真菌种属，目前被描述且被接受的有70余种。蜜环菌属真菌多数物种是木本和藤本植物根腐病的主要致病菌之一，防治较为困难，给林业、农业和园林带来巨大损失。但是，蜜环菌属的某些真菌也具有一定的经济价值，比如子实体的食用和药用价值。此外，蜜环菌属某些真菌应用于一些药用真菌和植物的栽培，如猪苓（Polyporus umbellatus）和天麻（Gastrodia elata）。天麻是一类以蜜环菌为寄主的菌异养植物，能利用蜜环菌为其提供营养完成自己的生活史。作为传统中药材，人工栽培于我国诸多地区，广泛应用于食品和医疗领域，创造了巨大的经济收益。由于天麻的块茎生长主要依赖蜜环菌提供营养，因此蜜环菌菌种的品质直接决定人工栽培天麻的产量与质量。然而，目前天麻栽培所用的蜜环菌菌种极为混乱，存在着菌种退化、菌种适应性差、菌种错误等诸多问题。此外，天麻组织培养的高难度导致其遗传操作的极大困难性，进展缓慢；相对而言，对蜜环菌进行遗传操作相对较为容易。蜜环菌的遗传转化系统在菌种退化与复壮、菌种遗传改良以及蜜环菌的生物防治等研究和应用中起重要作用，也为菌异养植物天麻和蜜环菌互作分子机制研究提供基础平台。大部分蜜环菌属真菌子实体的诱导困难，难以使用孢子进行遗传转化体系的构建。因此，在蜜环菌真菌菌丝体基础上构建遗传转化体系是一种可行性较高的选择。本论文主要使用栽培天麻的优良蜜环菌菌株A37（Armillaria sp.）的菌索作为实验材料，尝试使用原生质体和农杆菌介导的遗传转化方法构建蜜环菌遗传转化体系。主要研究结果如下：1、通过对培养基类型、光照以及温度的筛选，最终确定以葡萄糖、马铃薯汁、木屑汁为主的3号固体培养基作为蜜环菌A37的主要培养基。利用3号固体培养基，蜜环菌菌丝和菌索可以在23 ℃的暗培养条件下快速生长，为后续实验提供充足的材料。2、利用液体菌丝培养基培养蜜环菌菌丝，并作为实验材料来制备原生质体。但存在大量菌丝未被完全酶解、菌丝释放的原生质体数量少、破裂的情况。因此，原生质体的制备需要对酶解液的成分和比例、等渗溶液的pH以及盐浓度等进一步地优化。3、通过优化外源表达载体的启动子与终止子元件，使用eGFP与GUS作为报告基因，构建农杆菌介导的蜜环菌遗传转化体系。针对遗传转化效率较低的问题，以乙酰丁香酮（Acetosyringone）浓度、农杆菌OD值、侵染时间以及共培养时间等参数为对象，在蜜环菌A37中进行了遗传转化体系的优化，转化效率可达40%。4、利用构建的蜜环菌高效遗传转化体系，使用基因沉默（RNAi）技术对蜜环菌A37内源基因URA3成功地进行了遗传操作。; Armillaria, which is widely distributed worldwide, is a genus in the phylum Basidiomycota, comprising approximately 70 known species. Most Armillaria species cause root-rot disease in a wide variety of hosts, including woody and vine plants, and brings huge losses to forestry, agriculture, and gardening, and prevention and treatment are exceedingly difficult. However, some fungi of the genus Armillaria also have economic importance, such as edible and medical values. In addition, some species of the genus Armillaria are needed for cultivating medicinal fungi and plants, such as Polyporus umbellatus and Gastrodia elata.G. elata is a mycoheterotrophic plant, fully depending on Armillaria for providing nutrients. As a traditional Chinese medicine, it has been cultivated in many areas as a medicine as well as food fields in China. The growth and development of G. elata tuber mainly depend on Armillaria for providing nutrients, and therefore the quality of the Armillaria strains directly determines the yield and quality of cultivated G. elata. However, the Armillaria strains are extremely chaotic on the market, and there are many problems such as strain degradation, poor adaptability, and wrong strain. The difficult tissue culture has hampered the genetic modification of G. elata; by contrast, the genetic manipulation is relatively easy in Armillaria. The genetic transformation system may play an important role in prevention of strain degradation and strain rejuvenation, genetic improvement, and biological prevention and control for Armillaria, and also provides a basic platform for molecular study of the interaction between G. elata and Armillaria. It is difficult to induce fruiting bodies to gain spores in most Armillaria fungi; therefore a genetic transformation system using Armillaria spores is unfeasible. To establish a genetic transformation system in Armillaria, mycelium was chosen as the target. In this study, the strain, Armillaria sp. (A37), was used as the experimental material, and the protoplast and Agrobacterium tumefaciens mediated genetic transformation systems were attempted. The study mainly resulted in the following results：1. Through optimizing the culture media, light, and temperature, Armillaria A37 grew best when it was cultured in dark at 23 °C in medium NO. 3, which contains glucose, potato juice, and sawdust juice.2. Protoplasts were prepared using Armillaria hyphae cultured in liquid medium. However, we failed to obtain high quantity protoplasts. The preparation condition of Armillaria protoplast therefore need to be optimized, including the enzymes, isotonic solution, and pH value.3. Using eGFP and GUS as reporter genes, the Agrobacterium-mediated genetic transformation system has been established in Armillaria A37. The parameters of the system were optimized, including concentration of acetosyringone and Agrobacterium strain, time duration of infestation and co-culture. Finally, the transformation efficiency could reach 40%.4. Using the genetic transformation system, successful genetic manipulation of the endogenous gene URA3 in Armillaria A37 using gene silencing (RNAi).
语种	中文
	2022-06
学位授予单位	中国科学院大学
文献类型	学位论文
条目标识符	http://ir.kib.ac.cn/handle/151853/75092
专题	昆明植物所硕博研究生毕业学位论文
推荐引用方式 GB/T 7714	粟忠祥. 蜜环菌遗传转化体系的构建及应用[D]. 中国科学院大学,2022.

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